Proc. Natl. Acad. Sci. USAVol. 84, pp. 1921-1925, April 1987Cell Biology

Tubular lysosome morphology and distribution within macrophagesdepend on the integrity of cytoplasmic microtubules

(phorbol ester/10-nm filaments/cathepsin L/acid phosphatase)

JOEL SWANSON*, ANNE BUSHNELL, AND SAMUEL C. SILVERSTEINThe Rover Research Laboratory, Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, 630 West 168Street, New York, NY 10032

Communicated by Alex B. Novikoff, November 14, 1986 (received for review September 22, 1986)

ABSTRACT Pinocytosis of the fluorescent dye luciferyellow labels elongated, membrane-bound tubular organellesin several cell types, including cultured human monocytes,thioglycolate-elicited mouse peritoneal macrophages, and themacrophage-like cell line J774.2. These tubular structures canbe identified as lysosomes by acid phosphatase histochemistryand immunofluorescence localization of cathepsin L. Theabundance of tubular lysosomes is markedly increased bytreatment with phorbol 12-myristate 13-acetate (10 ng/ml).When labeled by pinocytosis of microperoxidase and examinedby electron microscopic histochemistry, the tubular lysosomeshave an outside diameter of -75 nm and a length of severalmicrometers; they radiate from the cell's centrosphere inalignment with cytoplasmic microtubules and intermediatefilaments. Incubation of phorbol myristate acetate-treatedmacrophages at 4°C or in medium containing 5 ,uM colchicineor nocodazole at 37°C leads to disassembly of microtubules andfragmentation of the tubular lysosomes. Return of the culturesto 37C or removal of nocodazole from the medium leads toreassembly of microtubules and the reappearance of tubularlysosomes within 10-20 min. We conclude that microtubulesare essential for the maintenance of tubular lysosome mor-phology and that, in macrophages, a significant proportion ofthe lysosomal compartment is contained within these tubularstructures.

Lysosomes generally are described as small spherical organ-elles (1). Observations of cultured cells indicate that althoughthis description is largely correct, lysosomes may assumeother shapes, either by conforming to ingested particles or byother intracellular forces. Buckley (2) observed thatlysosomes of cultured fibroblasts contained long, tubularextensions, which interconnected as a network. Others (3-5)have noted these elongated lysosomes as well. This paperexamines the role of cytoplasmic microtubules in shaping andpositioning these organelles in macrophages.

MATERIALS AND METHODSCells. Thioglycolate-elicited mouse peritoneal macro-

phages (thio-macrophages) were harvested from female ICRmice (Trudeau Institute, Saranac Lake, NY) as described (4),suspended in Eagle's minimal essential medium containing10% heat-inactivated fetal bovine serum (M1OF), and platedon 12-mm-diameter, no. 1 coverslips at 2 x 105 cells percoverslip for 2 hr at 37°C. Coverslips were washed to removenonadherent cells and maintained for up to 5 days in M1OF.

J774.2 macrophages, provided by J. Unkeless (Mt. SinaiSchool of Medicine, New York), were maintained in suspen-sion at 37°C in Dulbecco's modified minimal essential medi-um containing 10% heat-inactivated fetal bovine serum

(DM10F). For light microscopic observations, 105 cells wereplated on each 12-mm coverslip and incubated 24-48 hr.

Light Microscopy. Thio-macrophages or J774.2 cells oncoverslips were incubated 1-2 hr in medium containinglucifer yellow CH (1 mg/ml), washed in ice-cold PD (137 mMNaCl/3 mM KCl/7 mM phosphate, pH 7.4), returned to wellscontaining M1OF without lucifer yellow, and incubated 20-30min at 370C to allow dye efflux from the pinosome/endosomecompartment. Coverslips were inverted onto microscopeslides with M1OF (37°C) as the cells' medium. Macrophageswere observed live, using a Zeiss Photomicroscope equippedwith a 63x oil immersion lens (n.a. 1.4) and photographedusing Kodak Tri-X film (ASA 400).

Pinocytosis of fluorescein-labeled dextran (5 mg/ml) orTexas red-labeled ovalbumin (50 ,g/ml) was carried out inthe same manner as outlined above for lucifer yellow.

Immunofluorescence. Macrophages on coverslips wereprocessed for indirect immunofluorescence microscopy aftereither (a) no pretreatment, (b) incubation in phorbol 12-myristate 13-acetate (PMA)-containing medium, or (c) incu-bation in medium containing lucifer yellow with or withoutPMA. Cells were fixed for 45 min at 37°C in periodate/lysine/paraformaldehyde fixative (pH 7.4) (6) prepared just beforeuse, washed five times (3 min per wash) at room temperaturein phosphate-buffered saline containing Ca2' and Mg2+(PBS: 137mM NaCl/3 mM KCl/7mM phosphate, pH 7.4/0.5mM MgCl2/1 mM CaCl2), extracted in methanol (10-15 secat -20°C), washed twice more in PBS, and four more timesin PBS with 2% heat-inactivated goat serum (PBS/GS). Thislast solution was employed in all subsequent washings; allsubsequent processing was done at room temperature.Coverslips were applied cell-side-down onto 50-80 ,1u ofprimary antibody solutions, incubated 60 min, washed fivetimes (3 min each) in PBS/GS, incubated cell-side-downagain with secondary antibodies for 45 min, washed again,and mounted in 90% glycerol/10% PBS containing p-phenylenediamine (1 mg/ml) to reduce bleaching duringmicroscopic study (7). Little fluorescent staining was ob-served with secondary antibodies when primary antibodieswere omitted from the immunofluorescence protocol (datanot shown).

Electron Microscopy and Cytochemistry. Thio-macro-phages were plated at 6-7 x 105 peritoneal cells per well ofa 24-well culture dish. For localization of pinocytosedmicroperoxidase, cells were pretreated with medium con-taining PMA (10 ng/ml) and microperoxidase (1 mg/ml) for2 hr at 37°C, followed by 15 min in microperoxidase-free,PMA-containing solutions, and fixed at 37°C with 2% gluta-raldehyde in 0.1 M cacodylate buffer (pH 7.4) containing

Abbreviations: PMA, phorbol 12-myristate 13-acetate; thio-macro-phages, thioglycolate-elicited macrophages.*Present address: Department of Anatomy and Cellular Biology,Harvard Medical School, 25 Shattuck Street, Boston, MA 02115.

1921

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 16

, 202

2

Proc. Natl. Acad. Sci. USA 84 (1987)

4.5% sucrose. Cells were washed with 0.1 M cacodylatebuffer (pH 7.2), incubated with H202 and diaminobenzidinein Tris imidazole buffer (pH 8.6) as described (8), all at roomtemperature; and then washed and processed for electronmicroscopy as described (9). Sections, either unstained orcontrasted with uranyl acetate and lead citrate, were exam-ined with a JEOL 1200 EX electron microscope. Acidphosphatase was localized as described (10, 11), usingtrimetaphosphate (10) or CMP (11) as substrates.

Materials. Lucifer yellow CH (potassium salt) and Texasred-labeled ovalbumin were from Molecular Probes (Eugene,OR). PMA, colchicine, nocodazole, p-phenylenediamine,microperoxidase (MP-II), fluorescein-labeled dextran, sodi-um glycerophosphate, and paraformaldehyde were fromSigma. Lumicolchicine, a colchicine photoproduct that haslost microtubule-depolymerizing activity, was prepared asdescribed (12). Rhodamine-conjugated goat anti-rabbit IgGwas from Tago (Burlingame, CA). Fluorescein-conjugatedgoat anti-mouse IgG Fc fragment was from Cooper Biomed-ical (Malvern, PA). Rabbit antiserum against lysosomalmembrane glycoprotein lgpl20 (13), a gift from I. Mellman(Yale University), was stored in dimethyl sulfoxide at -200Cand used at a 1:300 dilution in PBS/GS. D. Portnoy(Rockefeller University) provided rabbit antibody againstcathepsin L; the antibody was raised and affinity-purifiedusing a recombinant fusion protein from Escherichia coli (14).Mouse monoclonal antibody against tubulin, clone B512 (15),was from G. Piperno (Rockefeller University).

RESULTSThe lysosomes of living thio-macrophages were displayed forfluorescence microscopy by allowing cells to pinocytose thedye lucifer yellow for 1-2 hr. The cells were then washed andincubated in lucifer yellow-free medium for 20-30 min toallow dye efflux from the pinosomal endosome compartment.Although most of the lysosomes were spherical or irregular,afew were long and thin (Fig. 1 A and B). The number ofthesetubular organelles was increased by treatment of thio-macrophages with PMA (Fig. 1 C and D). PMA enhanced cellspreading (16, 17) and stimulated extension of the fluorescenttubules to the cells' margins. At 37°C, the tubules were seenmoving in the cytoplasm. Smaller tubules moved freely, insaltatory movements characteristic of microtubule-basedmotility (18, 19). Longer tubules sometimes moved alongtortuous pathways in the cytoplasm but more often appearedto be linear and immobile. No general centrifugal or centri-petal flow was obvious.

FIG. 2. Lucifer yellow-labeled tubules of an unstimulated J774.2macrophage. Note the reticular and apparently interconnecting formof the tubular networks. (Bar = 5 ,m.)

Similar structures were abundant without PMA treatmentin J774.2 macrophages (Fig. 2) and in cultured humanmonocytes (data not shown). In resident peritoneal macro-phages ofmice, as well as in macrophages elicited by BacillusCalmette-Gudrin, tubules were elicited by treatment withPMA (data not shown). These structures also could belabeled by pinocytosis of fluorescein-labeled dextran orTexas red-conjugated ovalbumin (data not shown).Most fixation protocols employing formaldehyde or gluta-

raldehyde disrupted the tubules, producing linear arrays offluorescent vesicles similar to those observed by Phaire-Washington et al. (16). However, the use of glutaraldehydeat 37°C preserved tubule integrity. We used this fixationmethod to examine the shape and distribution of theseorganelles at the ultrastructural level. PMA-stimulated thio-macrophages were allowed to pinocytose microperoxidasefor 120 min to label the lysosomal system. The cells were thenprepared for cytochemical localization of microperoxidase(8) and sectioned parallel to the plane of the substrate. Thisprotocol provided many longitudinal sections of the tubularorganelles [outer diameter 75 + 24 nm (n = 22)] containing themicroperoxidase reaction product (Fig. 3). The reactionproduct was often separated from the tubule membrane by aclear area of uniform width (20 nm), which we interpret asglycocalyx (20) (Fig. 3 B-D).

FIG. 1. Intracellular fluorescence after pinocytosis of lucifer yellow by thio-macrophages. An unstimulated thio-macrophage is shown in A(phase-contrast) and B (fluorescence). C and D show a thio-macrophage after treatment for 60 min with PMA. Arrowheads indicate tubularlysosomes. (Bar = 5 im.)

1922 Cell Biology: Swanson et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 16

, 202

2

Proc. Natl. Acad. Sci. USA 84 (1987) 1923

: -

Ace BN

FIG. 3. Tubular lysosome labeled by pinocytosis of microperoxidase. Thio-macrophages were incubated 2 hr in medium with PMA andmicroperoxidase, then fixed and prepared for histochemical localization of the enzyme. (A-C) Longitudinal sections. Note the association ofvesicles with cytoplasmic microtubules (single arrowheads) in A and C. Reaction product is excluded from the luminal region immediatelyadjacent to the membrane (double arrowheads in B and C). The continuity of this clear zone along the length of the tubular lysosome shownin B indicates that this is a consistent feature of this organelle. (Bars = 5 A.m.) (D) Cross-section of tubular lysosomes. Arrowheads indicatecytoplasmic microtubules. (Bar = 0.5 aum.)

In many sections, the tubules appeared to have eitherfragmented into vesicles or wrinkled during fixation so as topass in and out of the section plane (Fig. 3 A and C). Thesevesicles were usually aligned with cytoplasmic microtubulesor intermediate filaments (ref. 16; Fig. 3 A-C; Fig. 5 C and D).These tubules were identified as lysosomes as follows.

First, they were labeled by pinocytosed substances includinglucifer yellow, fluorescein-conjugated dextran (Mr 4000),Texas red-conjugated ovalbumin, and, at the ultrastructurallevel, microperoxidase (Fig. 3). Second, they contained acidphosphatase (Fig. 4 and ref. 16). Third, the tubular or lineardistribution of pinocytosed lucifer yellow coincided with thedistribution of rhodamine-labeled antibodies against cathep-sin L (Fig. 5 A and B), and against the lysosomal membraneglycoprotein lgp120 (data not shown).

Formaldehyde-containing fixatives generally disrupted theintegrity of tubular lysosomes, but in many cells the presence

of tubules could be inferred from the linear distribution offluorescent organelles (Fig. 5). In some cells, such as PMA-treated resident macrophages, the tubules remained intactafter paraformaldehyde fixation.The alignment of tubular lysosomes and cytoplasmic mi-

crotubules shown in Fig. 3 suggested a functional associationof the two organelles. Immunofluorescence localization ofantibodies against tubulin and cathepsin L showed closecorrespondence between the orientation of tubular lyso-somes (preserved as rows of vesicles) and cytoplasmicmicrotubules (Fig. 5 C and D).To examine this relationship further, we incubated J774.2

cells or PMA-treated thio-macrophages in medium contain-ing lucifer yellow (to label lysosomes) and colchicine,lumicolchicine, or nocodazole. When live cells were exam-ined by fluorescence microscopy, the colchicine- ornocodazole-treated cells lacked tubular lysosomes (Fig. 6 A

Cell Biology: Swanson et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 16

, 202

2

1924 Cell Biology: Swanson et al.

FIG. 4. Acid phosphatase localization in tubules, using trimeta-phosphate as substrate. Similar results were obtained using CMP.(Bar = 1 ,um.)

and B). Fluorescent vesicles were circular or irregular andremained immobile in the cytoplasm (i.e., no saltatorymovement). Control or lumicolchicine-treated cells con-tained abundant tubular lysosomes (data not shown). Whennocodazole-treated thio-macrophages, which did not containvisible microtubules (Fig. 6D), were washed and incubated indrug-free medium, a condition that allows rapid repolymer-ization of microtubules (Fig. 6 E and F), tubular lysosomesreappeared within 15-20 min (Fig. 6C). Microtubule-depo-lymerizing drugs had similar effects on tubular lysosomemorphology in J774.2 macrophages (data not shown).

DISCUSSIONIn well-spread macrophages, tubular lysosomes often forman extensive reticulum, which is best appreciated by exam-ining several planes of focus. Perhaps, analogous to theendoplasmic reticulum, the lysosomal system of macro-phages is composed under some conditions ofa small numberof interconnecting compartments.The prevalence of tubular lysosomes in cells other than

macrophages remains to be determined. It is likely that theseorganelles have been missed by many observers, since thelow temperature used to wash and fix cells disrupts micro-tubules and tubular lysosomes, leaving only small vesicles orrows of vesicles (see ref. 16). Robinson et al. (3) preservedtubule structure by rapidly freezing their specimens. We havefound that a conventional glutaraldehyde fixative containingsucrose, when used at 37TC, is sufficient for electron micros-copy; for light microscopic immunocytochemistry, the fixa-tive of McLean and Nakane (6) sometimes preserves tubularlysosomes.With or without PMA, macrophages contain an abundance

of cytoplasmic microtubules. Therefore, the increase intubular lysosomes that accompanies PMA stimulation isprobably not due to a change in the number of microtubulesbut instead results from increased association betweenlysosomes and microtubules. The capacity ofPMA to inducetubular lysosome morphology indicates that the associationof lysosomes with microtubules can vary with the physio-logical state of the cell. In this regard, it is noteworthy thatJ774.2 macrophages, which constitutively display tubularlysosomes, have none during mitosis, a time when most ofthecell's microtubules are engaged in the spindle (J.S., unpub-lished observations of 20 mitotic cells). This suggests regu-lation of lysosome elongation by microtubules.While our results show that intact microtubules are nec-

essary for tubular lysosome movement and shape, they donot prove that there is a direct association between the twoorganelles. Both cytoplasmic microtubules and intermediatefilaments lie parallel to tubular lysosomes. Microtubule-depolymerizing drugs alter the distribution of both elements(16, 21). Thus, the observed disintegration of tubularlysosomes in nocodazole-treated cells could be a conse-quence of changes in distribution of either microtubules orintermediate filaments. Nevertheless, we think the bulk ofthe evidence favors a major role for microtubules in main-taining tubular lysosomes. Colchicine and nocodazole de-

FIG. 5. Immunofluorescence colocalization of lucifer yellow and cathepsin L in tubular lysosomes, and the association of these tubularlysosomes with cytoplasmic microtubules. (A) Lucifer yellow fluorescence. (B) Rhodamine fluorescence of secondary antibody (goat anti-rabbitIgG), which identifies sites of primary antibody binding (rabbit anti-cathepsin L). (C) Fluorescein-labeled goat anti-mouse IgG Fc fragment,which identifies binding sites of mouse monoclonal anti-tubulin antibody. (D) Rhodamine fluorescence, indicating indirect labeling of cathepsinL, as in B.

Proc. Natl. Acad Sci. USA 84 (1987)

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 16

, 202

2

Proc. Natl. Acad. Sci. USA 84 (1987) 1925

FIG. 6. Effects of microtubule-depolymerizing drugs on microtubules and tubular lysosome morphology. (A-C) Thio-macrophages wereincubated 60 min in medium (M10F, see Materials and Methods) containing lucifer yellow and PMA and then 60 min in M1OF with lucifer yellow,PMA, and 5 AM colchicine (A), 5 ,uM nocodazole (B), or nocodazole followed by 15 min in nocodazole-free medium (C). Cells were washedfree of extracellular lucifer yellow before observation; all but those in (C) remained in drug-containing medium during microscopic observation.Arrowheads inA designate the cell margins as determined by phase-contrast microscopy (not shown). InA and B, note that lysosomes containinglucifer yellow do not extend to the margins of the cell. (D-F) PMA-treated thio-macrophages incubated in medium with nocodazole for 60 min(D) or 60 min in medium with nocodazole followed by incubation in drug-free medium for 12 min (E) or 20 min (F). These cells were fixed andprocessed for immunofluorescence localization of tubulin. Fluorescence in D is diffuse (no microtubules), but microtubules reappear uponreincubation in drug-free medium (E and F). (Bars = 5 Am.)

polymerize microtubules but only redistribute intermediatefilaments. If intermediate filaments directly shapelysosomes, we might expect colchicine to relocate tubularlysosomes to the vicinity of the nucleus but not necessarilyto cause their disintegration. At present we consider themicrotubule as an armature that maintains the tubular struc-ture of lysosomes.

We gratefully acknowledge the generous support of Mr. SamuelRover. We also thank Eugene Burke and Kevin Costello for technicalassistance; Drs. Ira Mellman, Eugenia Wang, Gianni Piperno, andDaniel Portnoy for generously supplying us with antibodies; and Drs.Phyllis and Alex Novikoff for assistance with the acid phosphatasecytochemistry. This research was funded in part by National Re-search Service Award F32 AI06880 (to J.S.) and Public HealthService Grant AI20516 (to S.C.S.).

1. Bainton, D. F. (1981) J. Cell Biol. 91, 66s-76s.2. Buckley, I. K. (1973) Lab. Invest. 29, 411-421.3. Robinson, J. M., Okada, T., Castellot, J. J. & Karnovsky,

M. J. (1986) J. Cell Biol. 102, 1615-1622.4. Swanson, J. A., Yirinec, B. D. & Silverstein, S. C. (1985) J.

Cell Biol. 100, 851-859.

5. Novikoff, P. M. (1986) Advances in Cell Biology, ed. Miller,K. (Jai, Greenwich, CT), in press.

6. McLean, I. W. & Nakane, P. K. (1974) J. Histochem.Cytochem. 22, 1077-1083.

7. LeDizet, M. & Piperno, G. (1986) J. Cell Biol. 103, 13-22.8. Simionescu, N., Simionescu, M. & Palade, G. E. (1975) J. Cell

Biol. 64, 586-607.9. Swanson, J., Yirinec, B., Burke, E., Bushnell, A. & Silver-

stein, S. C. (1986) J. Cell. Physiol. 128, 195-201.10. Oliver, C. (1980) J. Histochem. Cytochem. 28, 78-81.11. Novikoff, A. B. & Novikoff, P. M. (1977) Histochem. J. 9,

525-551.12. Wilson, L. & Friedkin, M. (1966) Biochemistry 5, 2463-2468.13. Lewis, V., Green, S. A., Marsh, M., Vihko, P., Heleneius, A.

& Mellman, I. (1985) J. Cell Biol. 100, 1839-1847.14. Portnoy, D. A., Erickson, A. H., Kochan, J., Ravetch, J. V.

& Unkeless, J. C. (1986) J. Biol. Chem. 261, 14697-14703.15. Piperno, G. & Fuller, M. T. (1985) J. Cell Biol. 101, 2085-2094.16. Phaire-Washington, L., Silverstein, S. C. & Wang, E. (1980) J.

Cell Biol. 86, 641-655.17. Phaire-Washington, L., Wang, E. & Silverstein, S. C. (1980) J.

Cell Biol. 86, 634-640.18. Freed, J. J. & Lebowitz, M. M. (1970) J. Cell Biol. 45,

334-354.19. Schroer, T. A. & Kelly, R. B. (1985) Cell 40, 729-730.20. Neiss, W. F. (1984) Histochemistry 80, 603-608.21. Goldman, R. D. (1971) J. Cell Biol. 51, 752-762.

Cell Biology: Swanson et al.

Dow

nloa

ded

by g

uest

on

Feb

ruar

y 16

, 202

2